Preparation of cephem and isooxacephem derivatives

ABSTRACT

The invention is concerned with a new process for the preparation of compounds of formula ##STR1## wherein R 1  is trityl, acetyl, tetrahydropyranyl or cyclopentyl; 
     R 2  is hydrogen, hydroxy, lower alkyl, cycloalkyl, lower alkoxy, lower alkenyl, lower alkynyl, aryl, aryloxy, aryl-lower alkyl, aryl-lower alkoxy or heterocyclyl or heterocyclyl-lower alkyl; the lower alkyl, cycloalkyl, lower alkoxy, lower alkenyl, cycloalkenyl, lower alkynyl, aryl-lower alkyl, aryl, aryloxy, aryl-lower alkoxy, the heterocyclyl moieties being unsubstituted or substituted with at least one group selected from carboxy, amino, nitro, cyano, lower alkyl, lower alkoxy, hydroxy, halogen, --CONR 21  R 22 , --N(R 22 )COOR 23 , R 22  CO--, R 22  OCO-- or R 22  COO--, wherein R 21  is hydrogen, lower alkyl, or cycloalkyl; R 22  is hydrogen or lower alkyl; R 23  is lower alkyl, lower alkenyl or a carboxylic acid protecting group; 
     Y is --S-- and Z is --CH 2  -- or 
     Y is --CH 2  -- and Z is --O--, 
     by acylation of a compound of formula ##STR2## with an activated carboxylic acid derivative of formula ##STR3## wherein R 3  is lower alkyl, and R 1 , R 2 , X, Y, Z have the significance given above; and it is further concerned with compounds of formula III.

This is a divisional of U.S. Ser. No. 08/856,070, filed May 14, 1997 nowU.S. Pat. No. 5,883,247.

FIELD OF THE INVENTION

This invention relates to a process for preparing cephem andisooxacephem cephalosporin derivatives, and intermediates therefor.

BACKGROUND OF THE INVENTION

U.S. Pat. No. 5,523,400, issued Jun. 4, 1996 (Wei, et al) disclosescertain cephalosporin antibiotics having a lactam moiety at the 3position of the cephalosporin.

U.S. Pat. No. 4,409,214, issued Oct. 11, 1983 (Takaya, et al) discloses7-acylamino-3-vinyl-cephalosporanic acid derivatives havingantimicrobial activity.

Kamachi, et al, J. Antibiotics (December 1990) Vol. 43, pages 1564-1572discloses the synthesis of 1-Acetoxyethyl 7-(Z)-2-(2-aminothiazol4-yl)-2-hydroxyiminoacetamido!-3-(Z)-1-propenyl!-3-cephem4-carboxylate by acylation of the cephem unitwith an activated aminothiazole derivative in which the activating groupis a benzothiazolyl ester.

European Patent Publication No. EP 620228, published Oct. 19, 1994(Lucky, Ltd.) discloses thiophosphate derivatives of thia(dia)zoleacetic acid for use in the preparation of β-lactam antibiotics. In thiscase the cephem unit is acylated using an activated aminothiazolederivative in which the activating group is a mixed anhydride ofthiophosphoric acid.

Other acyl groups which can be used to acylate β-lactam antibiotics maybe found in Cephalosporins and Penicillins, Flynn, ed, Academic Press(1972); Belgian Patent No. 866,038, published Oct. 17, 1978; BelgianPatent No. 867,994, published Dec. 11, 1978; and U.S. Pat. No.3,971,778, issued Jul. 27, 1976.

SUMMARY OF THE INVENTION

The present invention is concerned with a process for the preparation ofcephem- and isooxacephem derivatives of formula ##STR4## wherein R¹ istrityl, acetyl, tetrahydropyranyl or cyclopentyl;

R² is hydrogen, hydroxy, lower alkyl, cycloalkyl, lower alkoxy, loweralkenyl, lower alkynyl, aryl, aryloxy, aryl-lower alkyl, aryl-loweralkoxy or heterocyclyl or heterocyclyl-lower alkyl; the lower alkyl,cycloalkyl, lower alkoxy, lower alkenyl, cycloalkenyl, lower alkynyl,aryl-lower alkyl, aryl, aryloxy, aryl-lower alkoxy, the heterocyclylmoieties being unsubstituted or substituted with at least one groupselected from carboxy, amino, nitro, cyano, lower alkyl, lower alkoxy,hydroxy, halogen, --CONR²¹ R²², --N(R²²)COOR²³, R²² CO--, R²² OCO-- orR²² COO--, wherein R²¹ is hydrogen, lower alkyl, or cycloalkyl; R²² ishydrogen or lower alkyl; R²³ is lower alkyl, lower alkenyl or acarboxylic acid protecting group;

Y is --S-- and Z is --CH₂ -- or

Y is --CH₂ -- and Z is --O--,

by acylation of a compound of formula ##STR5## with an activatedcarboxylic acid derivative of formula ##STR6## wherein R³ is loweralkyl, and R¹, R², Y, Z have the significance given above.

The compounds of formula I are useful as antibiotics having potent andbroad antibacterial activity.

This invention provides compounds of formula III.

As used herein, the term "lower alkyl" refers to both straight andbranched chain saturated hydrocarbon groups having 1 to 8, preferably 1to 4 carbon atoms, for example, methyl, ethyl, n-propyl, isopropyl,tertiary butyl and the like.

By the term "cycloalkyl" is meant a 3-7 membered saturated carbocyclicring e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and thelike.

The term "lower alkoxy" refers to an oxygen radical having an alkylgroup as defined above, examples include methoxy, ethoxy, n-propyloxyand the like.

As used herein, "lower alkenyl" refers to an unsubstituted orsubstituted hydrocarbon chain radical having from 2 to 8 carbon atoms,preferably from 2 to 4 carbon atoms, and having at least one olefinicdouble bond, e.g. vinyl, allyl and the like.

As used herein, "lower alkynyl" refers to an unsubstituted orsubstituted hydrocarbon chain radical having from 2 to 8 carbon atoms,preferably 2 to 4 carbon atoms, and having at least one triple bond.

The term "halogen" used herein refers to all four forms, that ischlorine or chloro; bromine or bromo; iodine or iodo; and fluorine orfluoro.

By the term "aryl" is meant a radical derived from an aromatichydrocarbon by the elimination of one atom of hydrogen and can besubstituted or unsubstituted. The aromatic hydrocarbon can bemononuclear or polynuclear. Examples of aryl radicals of the mononucleartype include phenyl, tolyl, xylyl, mesityl, cumenyl, and the like.Examples of aryl radicals of the potynuclear type include naphthyl,anthryl, phenanthryl, and the like. The aryl group can have at least onesubstituent selected from, as for example, halogen, hydroxy, cyano,carboxy, carbamoyl, nitro, amino, aminomethyl, lower alkyl, lower alkoxyor trifluormethyl. Examples include 2-fluorophenyl, 3-nitrophenyl,4-nitrophenyl, 4-methoxyphenyl, 4-hydroxyphenyl and the like.

By the term "aryl-lower alkyl" is meant a lower alkyl group containingan aryl group as defined above, for example benzyl.

As used herein, "aryloxy" is an oxygen radical having an arylsubstituent as defined above (i.e., --O-aryl).

As used herein, "aryl-lower alkoxy" is an oxygen radical having anaryl-lower alkyl substituent. (i.e., --O-lower-alkyl-aryl).

As used herein, "heterocyclyl ring" refers to the residue of anunsaturated or saturated, unsubstituted or substituted 5-, 6-, or7-membered heterocyclic ring containing at least one hetero atomselected from the group consisting of oxygen, nitrogen, or sulfur.Exemplary heterocyclyl groups include, but are not limited to, e.g., thefollowing groups: pyridyl, pyridiniumyl, pyrazinyl, piperidyl,piperidino, N-oxido-pyridyl, pyrimidyl, piperazinyl, pyrrolidinyl,pyridazinyl, N-oxide-pyridazinyl, pyrazolyl, triazinyl, imidazolyl,thiazolyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl,1,2,5-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl,1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl,1H-tetrazolyl, 2H-tetrazolyl, thienyl, furyl, hexamethyleneiminyl,oxepanyl, 1H-azepinyl, thiophenyl, tetrahydrothiophenyl,3H-1,2,3-oxathiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadithiolyl,isoxazolyl, isothiazolyl, 4H-1,2,4-oxadiazinyl, 1,2,5-oxathiazinyl,1,2,3,5-oxathiadiazinyl, 1,3,4-thiadiazepinyl, 1,2,5,6-oxatriazepinyl,oxazolidinyl, tetrahydrothienyl, and others. Substituents for theheterocyclic ring include lower-alkyl, lower-alkoxy, halogen,trifluoromethyl, trichloroethyl, amino, mercapto, hydroxy, carboxy orcarbamoyl. Preferred examples of substituted heterocyclyl groups aremono-substituted and include 5-methyl-isoxazol-3-yl,N-methyl-pyridinium-2yl, 1-methyl-tetrazolyl and the like.

As used herein, "heterocyclyl-lower alkyl" refers to a lower alkyl groupcontaining a heterocyclic group as defined above, e.g.tetrazolyl-methyl, tetrahydrofuranyl-methyl, thiophenyl-methyl orbenzimidazolyl-methyl.

The heterocyclic ring can also be substituted by an optionallysubstituted phenyl ring such as 2,6-dichlorophenyl. Preferred is2,6-dichlorophenyl-5-methyl-isoxazolyl.

A further substituent of the heterocyclic ring is oxo, such as in2-oxo-oxazolidin-3-yl, 1,1-dioxo-tetrahydrothien-3-yl.

The heterocyclic ring can also be fused together with a benzene ring.

By the term "substituted phenyl" is meant phenyl mono or di-substituted.

The term "carboxylic acid protecting group" refers to protecting groupsconventionally used to replace the acidic proton of a carboxylic acid.Examples of such groups are described in Greene, T., Protective Groupsin Organic Synthesis, Chapter 5, pp. 152-192 (John Wiley and Sons, Inc.1981), incorporated herein by reference. These examples includemethoxymethyl, methylthiomethyl, tetrahydropyranyl, tetrahydrofuranyl,methoxyethoxymethyl, benzyloxymethyl, phenacyl, p-bromophenacyl,α-methylphenacyl, p-methoxyphenacyl, diacylmethyl, N-phthalimidomethyl,ethyl, 2,2,2-trichloroethyl, 2-haloethyl, ω-chloroalkyl,2-(trimethylsilyl)ethyl, 2-methylthioethyl,2-(p-nitrophenyl-sulfenyl)ethyl, 2-(p-toluenesulfonyl)ethyl,1-methyl-1-phenylethyl, t-butyl, cyclopentyl, cyclohexyl, allyl,cinnamyl, phenyl, p-methylthiophenyl, benzyl, triphenylmethyl,diphenylmethyl, bis(o-nitrophenyl)methyl, 9-anthrylmethyl,2-(9,10-dioxo)anthrylmethyl, 5-dibenzosuberyl, 2,4,6-trimethylbenzyl,p-bromobenzyl, o-nitrobenzyl, p-nitrobenzyl, p-methoxybenzyl, piperonyl,4-picolyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,i-propyl-dimethylsilyl, phenyldimethylsilyl, S-t-butyl, S-phenyl,S-2-pyridyl, N-hydroxypiperidinyl, N-hydroxysuccinimidoyl,N-hydroxyphthalimidoyl, N-hydroxybenzo-triazolyl, O-acyl oximes,2,4-dinitrophenylsulfenyl, 2-alkyl-1,3-oxazolines,4-alkyl-5-oxo-1,3-oxazolidines, 5-alkyl-4-oxo-1,3-dioxolanes,triethylstannyl, tri-n-butylstannyl; the amides or hydrazides ofN,N-dimethylaminio, pyrrolidinyl, piperidinyl, o-nitrophenyl,7-nitroindolyl, 8-nitrotetrahydroquinolyl, p-benzenesulfonamide,hydrazides, N-phenylhydrazide, N,N'-diisopropylhydrazide. Preferred arebenzyhydryl, t-butyl, p-nitrobenzyl, p-methoxybenzyl and allyl.

DETAILED DESCRIPTION OF THE INVENTION

The process according to the invention is especially suited for thepreparation of cephem and isooxacephem derivatives respectively offormula I wherein R¹ is hydrogen, i.e. with an hydroxyimino group, whichhas to be protected during the acylation step. It is essential that theprotecting groups are cheap, easily removeable, recycleable and that noadditional purification steps are involved due to contamination of acatalyst used during the protecting and deprotecting process.Furthermore the protecting group should not interfer with the acylationstep.

It has been found that the acylation process according to invention isespecially suited for the acylation of cephem- and isooxacephemderivatives of formula II with an aminothiazol derivative of formula IIIwhich is activated as mixed anhydride of thiophosphoric acid and R¹ isprotected by a trityl, acetyl or tetrahydropyranyl group, preferably atrityl group. The yield of this reaction as well as the purity of theproduct are excellent and the protecting groups are easily removed toyield hydroxyimino compounds, i.e. compounds of formula I wherein R¹ ishydrogen.

The acylation of a compound of formula II with the activated compound offormula III is preferably carried out in a polar solvent as dimethylformamide (DMF), dichloromethane, or a mixture of DMF/i-pronanol/waterin presence of a base as e.g. triethylamine, at a temperature of about-10° C. to about 60°, preferably from about 0° C. to about 30° C.

The compounds of formula III are part of the present invention. They canbe prepared as follows.

To obtain (Z)-(2-aminothiazol-4-yl)-trityl (or acetoxy,tetrahydropyranyl or cyclopentyl)oxyimino acetic acid, their precursorthe unprotected (Z)-(2-aminothiazol-4-yl)oxyimino acetic acid ethylester(compound A), is commercially available. This compound is then protectedas follows:

a) For the preparation of the trityl derivative (as used in example 1)the compound A is deprotonated and treated with tritylchloride to form(Z)-2-(aminothiazol4-yl)trityl-oxyimino acetic acid ethylester which isthen hydrolysed to yield the free acid.

b) For the preparation of the acetyl derivative (as used in example 2)compound A is hydrolysed to form the free acid(Z)-2-(aminothiazol4-yl)oxyimino acetic acid and subsequently treatedwith acetanhydride in the presence of potassium carbonate to form theacetyl derivative.

c) The tetrahydropyranyl derivative (as used in Example 3) is preparedby treating the glyoxylic acid derivative described below withO-(tetrahydro-pyran-2-yl)-hydroxyl-amine in the presence oftriethylamine in ethanol as depicted below: ##STR7## d) For thepreparation of the cyclopentyl derivative the compound A is deprotonatedand treated with cyclopentylbromide to form(Z)-2-(aminothiazol-4-yl)cyclopentyl-oxyimino acetic acid ethylesterwhich is then hydrolysed to yield the free acid. The free acid is thenreacted in analogy to example 1 to yield the activated acid.

The compounds of formula III are prepared by reaction of(Z)-(2-aminothiazol4-yl)-trityl (or acetoxy, or tetrahydropyranyl, orcyclopentyl) oxyimino acetic acid with di-lower alkylchloro thiophosphate in an organic solvent in the presence of a tert. amine. Thecompounds of formula m precipitate directly from the reaction mixture.Preferred tert. amine compounds are DABCO, tributylamine and mixturesthereof. The organic solvent is preferably dichloromethane ordimethylacetamide.

Compounds of formula II in which Y is S may be obtained from 3-cephemaldehyde as described in U.S. Pat. No. 5,523,400, issued Jun. 4, 1996(Wei et al).

Compounds of formula II in which Z is O may be obtained from3-isooxacephem aldehyde as shown in Scheme 1. ##STR8##

SCHEME 1

Wittig Reaction 1 to 3

The reaction of known 3-isooxacephem aldehyde (1) wherein the7-amino-protecting group is allyloxycarbonyl and the carboxy protectinggroup is allyl with a Wittig reagent (2) yields the coupling product(3). The reaction is carried out in the presence of a base which iseither an inorganic base (sodium or potassium hydroxide, sodium orpotassium carbonate etc.), an organic base (tertiary amines), anorganolithium compound such as butyl lithium or phenyl lithium or anepoxide such as 1,2-butyleneoxide. The preferred solvents are in thecase of inorganic base being used, water and water-miscible solvents(acetone, tetrahydroftiran, or alcohols etc.); in the case of organicbase being used, an inert solvent such as methylene chloride,chloroform, benzene, tetrahydrofuran; in the case of organolithium beingused, benzene or tetrahydrofuran, and in the case an epoxide being used,the epoxide itself (e.g. 1,2-butyleneoxide). The temperature for thereaction ranges from -20° C. to 80° C.

In the normal Wittig Reaction according to scheme 1, the E isomer is thepredominant product. Invariably, less than 10% Z-isomer is formed, theamount depending on the reagents and conditions.

The making of the Wittig reagent (2) can be carried out in a mannerknown per se; for example, by cyclization of a N-substituted dibromideusing a catalyst like Dowex as described in the European PatentApplication EPA 0 620 255.

Deprotection 3 to 4

The carboxylic acid protecting group R^(h) and the amino protectinggroup R^(f) are removed and the reaction conditions used are dependingon the nature of the protecting groups.

In the case of the amino protecting group being allyloxycarbonyl and thecarboxy protecting group being the allyl ester, Pd(0) generated in situis employed. In the case of the amino protecting group beingt-butoxycarbonyl and the carboxy protecting group being benzhydryl,trifluoroacetic acid is employed, at temperature of about -20° C. toabout room temperature.

Conventional carboxylic acid protecting groups and amino protectinggroups are described in Greene, T., Protective Groups in OrganicSynthesis, Chapter 5, pp. 152-192 (John Wiley and Sons, Inc. 1981).

The products in accordance with the invention can be used asmedicaments, for example, in the form of pharmaceutical preparations forenteral (oral) administration. The products in accordance with theinvention can be administered, for example, perorally, such as in theform of tablets, coated tablets, dragees, hard and soft gelatinecapsules, solutions, emulsions or suspensions, or rectally, such as inthe form of suppositories.

Pharmaceutical compositions containing these compounds can be preparedusing conventional procedures familiar to those skilled in the art, suchas by combining the ingredients into a dosage form together withsuitable, nontoxic, inert, therapeutically compatible solid or liquidcarrier materials and, if desired, the usual pharmaceutical adjuvants.

It is contemplated that the compounds are ultimately embodied intocompositions of suitable oral or parenteral dosage forms. Thecompositions of this invention can contain, as optional ingredients, anyof the various adjuvants which are used ordinarily in the production ofpharmaceutical preparations. Thus, for example, in formulating thepresent compositions into the desired oral dosage forms, one may use, asoptional ingredients, fillers, such as coprecipitated aluminumhydroxide-calcium carbonate, dicalcium phosphate or lactose;disintegrating agents, such as maize starch; and lubricating agents,such as talc. calcium stearate, and the like. It should be fullyunderstood, however, that the optional ingredients herein named aregiven by way of example only and that the invention is not restricted tothe use hereof. Other such adjuvants, which are well known in the art,can be employed in carrying out this invention.

Suitable as such carrier materials are not only inorganic, but alsoorganic carrier materials. Thus, for tablets, coated tablets, drageesand hard gelatine capsules there can be used, for example, lactose,maize starch or derivatives thereof, talc, stearic acid or its salts.Suitable carriers for soft gelatine capsules are, for example, vegetableoils, waxes, fats and semi-solid and liquid polyols (depending on thenature of the active substance; no carriers are, however, required inthe case of soft gelatine capsules). Suitable carrier materials for thepreparation of solutions and syrups are, for example, water, polyols,saccharose, invert sugar and glucose. Suitable carrier materials forsuppositiories are, for example, natural or hardened oils, waxes, fatsand semi-liquid or liquid polyols.

As pharmaceutical adjuvants there are contemplated the usualpreservatives, solubilizers, stabilizers, wetting agents, emulisifiers,sweeteners, colorants, flavorants, salts for varying the osmoticpressure, buffers, coating agents and antioxidants.

The compounds of formula I and their salts, or hydrates, can preferablybe used for parenteral administration, and for this purpose arepreferably made into preparations as lyophilisates or dry powders fordilution with customary agents, such as water or isotonic common saltsolution.

Depending on the nature of the pharmacologically active compound thepharmaceutical preparations can contain the compound for the preventionand treatment of infectious diseases in mammals, human and non-human, adaily dosage of about 10 mg to about 4000 mg, especially about 50 mg toabout 3000 mg, is usual, with those of ordinary skill in the artappreciating that the dosage will depend also upon the age, conditionsof the mammals, and the kind of diseases being prevented or treated. Thedaily dosage can be administered in a single dose or can be divided overseveral doses. An average single dose of about 50 mg, 100 mg, 250 mg,500 mg, 1000 mg, and 2000 mg can be contemplated.

The following examples illustrate the invention in more detail and arenot intented to be a limitation in any manner.

The following abbreviations were used:

mp melting point

HPLC high performance liquid chromatography

HPLC-analysis were performed as follows:

Sample preparation: The heterogeneous reaction mixture was dissolvedwith a little DMSO and diluted with CH₃ CN.

Instrument: HP-1050 HPLC System.

Column: Machery-Nagel Nucleosil 100-5 C18 AB, 250×4 mm.

Column temperature: 50° C.

Mobile Phase: A water+5% CH₃ CN; C CH₃ CN; D 0.03M potassium phosphatebuffer pH 3+10% CH₃ CN.

Gradient (t min!, A:C:D): (0, 85:0:15); (8, 15:70:15); (19, 15:70:15);(19.5, 85:0:15).

Flow: 1.2 ml/min.

Detection: UV 225 nm.

EXAMPLE 1

a) Preparation of (Z)-(2-Aminothiazol-4-yl)-trityloxyiminoacetic aciddiethoxythiophosphoryl ester ##STR9##

To a stirred suspension of 50 g(Z)-(2-aminothiazol4-yl)-trityloxyiminoacetic acid (116.4 mmol) and 130mg 1,4-diazabicyclo 2.2.2!octane (DABCO) (1.164 mmol) in 500 mldichloromethane was added under argon atmosphere 36 ml tributylamine(151 mmol). After 5 min, the red solution was cooled to 2° C. With theaid of a syringe pump was added over 30 min 24.5 ml diethylchlorothiophosphate (151 mmol). Stirring was continued at 2° C. for 1.5h. After approximately 30 min, the activated ester(Z)-(2-Aminothiazol-4-yl)-trityloxyiminoacetic aciddiethoxythiophosphoryl ester started to crystallize from the brownreaction mixture. The reaction was followed by HPLC. After 1 h, thestarting material was consumed. To the heterogeneous reaction mixturewas added dropwise over 1.5 h 750 ml water (to remove water solubleby-products) and over 40 min 500 ml n-hexane (to drive the precipitationof the product to completion). The suspension was stirred for 1 h at 2°C. and then filtered. The crystalline product was washed with 3×100 mlwater and 3×100 ml n-hexane/dichloromethane 3:1 and dried to constantweight. Activated ester (Z)-(2-Aminothiazol-4-yl)-trityloxyiminoaceticacid diethoxythiophosphoryl ester was obtained as a tan solid (64.24 g,yield=94.9%, HPLC=97.5 area %, mp=146° C.) and was stored under Ar at 4°C. No further purification was necessary and the product was used asisolated for the next step.

IR (KBr) 3444, 3092, 2983, 1770, 1618, 1541, 1490, 1024, 720; ¹ H-NMR(250 MHz, CDCl₃) δ 1.29 (dt,J₁ =7,J₂ =0.8,6H); 4.19 (dq,J₁ =8.0,J₂=7.0,4H); 6.01 (s,br,2H); 6.59 (s,1H); 7.26-7.34 (m,15H); ³¹ P-NMR (100MHz, CDCl₃) δ 59,05; ISP-MS 582.4 (100, M+H!⁺); MA calculated for C₂₈H₂₈ N₃ O₅ PS₂ C 57.82, H 4.85, N 7.22, S 11.02, P 5.33; found C 58.09, H4.96, N 7.21, S 10.92, P 5.35 and 0.35% water.

b) (6R, 7R)-7-(Z)-2-(2-Aminothiazol-4-yl)-2-trityloxyimino-acetylamino)!-3-(E)-1-cyclopropylmethyl-2-oxo-pyrrolidin-3-ylidenemethyl!-8-oxo-5-thia-1-azabicyclo4.2.0!oct-2-ene-2-carboxylic acid triethylammonium salt ##STR10##

To a stirred suspension of 22.78 g(E)-(6R,7R)-7-amino-3-(1-cyclopropylmethyl-2-oxo-pyrrolidin-3-ylidenemethyl)-8-oxo-5-thia-1-azabicyclo4.2.0!oct-2-ene-2-carboxylic acid (65.2 mmol) in 160 mldimethylformamide was added under argon 9.1 ml triethylamine (65.2 mmol)at 10° C. After 30 min, to the solution was added 48 ml 2-propanol and3.9 ml water causing the starting material to precipitate partially. Thesuspension was cooled to 2° C. and over 5 min was added in portions36.68 g activated ester (Z)-(2-Aminothiazol-4-yl)-trityloxyiminoaceticacid diethoxythiophosphoryl ester (66.5 mmol). Stirring was continued atroom temperature with exclusion of light for 17 h. The reaction wasfollowed by HPLC. To the slightly turbid reaction mixture was added over2 min 9.2 ml triethylamine (65.2 mmol, 1.0 eq) resulting in a clear,yellow solution. Reference material was added and after ca. 15 min, thereaction mixture became turbid, indicating the onset of crystallization.Stirring at room temperature was continued for 60 min and then 330 mlethylacetate was added dropwise over 90 min. To drive crystallization tocompletion the suspension was cooled to 2° C. and stirred for 3 h atthis temperature. The suspension was filtered. The crystalline productwas washed with 3×100 ml ice-cold ethylacetate and dried to constantweight. The cephalosporin (6R, 7R)-7-(Z)-2-(2-Aminothiazol-4-yl)-2-trityloxyimino-acetylamino)!-3-(E)-1-cyclopropylmethyl-2-oxo-pyrrolidin-3-ylidenemethyl!-8-oxo-5-thia-1-aza-bicyclo4.2.0!oct-2-ene-2-carboxylic acid triethylammonium salt was obtained asan off-white solid (51.56 g, yield=77%, HPLC=100 area %) and was storedunder Ar at 4° C. No further purification was necessary and the productwas used as isolated for the next step.

Anal.

¹ H-NMR (250 MHz, DMSO) δ 0.20 (m,2H); 0.46 (m,2H); 0.92 (m,1H); 3.14(d,J=7.0,2H); 3.22-4.09 (mm,7H); 3.78, 3.82 (2d,J=16.0,2H); 5.16(d,J=5.0,1H); 5.87 (dd,J₁ =13.2,J₂ =8.3,1H); 6.61 (s,1H); 7.23-7.33(mm,16H); 9.90 (d,J=8.3,1H)+signals for NEt₃ and DMF; calculated for C₄₀H₃₆ N₆ O₆ S₂ :C₆ H₁₅ N:C₃ H₇ NO=1:1:2 and 0.36% H₂ O C 61.94, H 6.50, N12.50, S 6.36; found C 61.49, H 6.29, N 12.17, S 6.69.

EXAMPLE 2

a) Preparation of (Z)-(2-Aminothiazol-4-yl)-acetoxyiminoacetic aciddiethoxythiophosphoryl ester ##STR11##

To a stirred solution of 134.9 g(Z)-(2-aminothiazol4-yl)-acetoxyiminoacetic acid dihydrate (508.6 mmol)and 570 mg 1,4-diazabicyclo 2.2.2!octane (DABCO) (5.09 mmol) in 1500 mldimethylacetamide was added under argon 158 ml tributylamine (661 mmol).The yellowish solution was cooled to -20° C. and over 30 min was addeddropwise 104 ml diethyl chlorothiophosphate (661 mmol). Stirring wascontinued at -20° C. for 3.5 h. The reaction was followed by HPLC. After3 h, all starting material was consumed. The reaction mixture wasallowed to warm up to 0° C. and over 1.0 h was added dropwise 2200 mlwater. The precipitated product was filtered, washed with water anddissolved in 800 ml dichloromethane. The aqueous layer wasback-extracted with 300 ml dichloromethane. The combined organic layerswere dried over 70 g sodium sulfate and concentrated under reducedpressure until the product started to crystallize. The residual solutionwas cooled to 2° C. and 1200 ml n-hexane was added dropwise over 1 h.The resulting suspension was stirred for 1 h at 2° C. and then filtered.The crystalline product was washed with n-hexane and dried to constantweight. (Z)-(2-aminothiazol-4-yl)-acetoxyiminoacetic aciddiethoxythiophosphoryl ester was obtained as a white solid (166.9 g,yield=86%, mp 128-130° C. and was stored under argon at -20° C. Nofurther purification was necessary and the product was used as isolatedfor the next step.

IR (KBr) 3429, 3260, 3172, 3135, 1795, 1770, 1619, 1538, 1174, 1020; ¹H-NMR (250 MHz, CDCl₃) δ 1.38 (dt,J₁ =7.0,J₂ =0.9,6H); 2.26 (s,3H); 4.34(dq,J₁ =8.0,J₂ =7.0,4H); 6.94 (s,1H); 7.50 (s,br,2H); ³¹ P-NMR (100 MHz,CDCl₃) δ 59.27; ISP-MS 404.1 (31, M+Na!⁺), 382.1 (100, M+H!⁺); MAcalculated for C₁₁ H ₁₆ O₆ N₃ PS₂ C 34.64, H 4.23, N 11.02, S 16.81, P8.12; found C 34.64, H 4.18, N 11.07, S 16.67, P 8.02.

b) Preparation of (6R,7R)-7-(Z)-2-(2-aminothiazol-4-yl)-2-acetoxyimino-acetylamino)!-3-(E)-1-cyclopropylmethyl-2-oxo-pyrrolidin-3-ylidenemethyl!-8-oxo-5-thia-1-azabicyclo4.2.0!oct-2-ene-2-carboxylic acid ##STR12##

Under an argon atmosphere to a stirred suspension of 25.6 g(E)-(6R,7R)-7-Amino-3-(1-cyclopropylmethyl-2-oxo-pyrrolidin-3-ylidenemethyl)-8-oxo-5-thia-1-azabicyclo4.2.0!oct-2-ene-2-carboxylic acid (73.3 mmol) in 120 mldimethylformamide was added 20 ml triethylamine (143 mmol) at 10° C.After 15 min, the solution was cooled to 0° C. and 28.5 g(Z)-(2-aminothiazol-4-yl)-acetoxyiminoacetic acid diethoxythiophosphonyl ester (74.8 mmol) was added in portions over 5 min.Stirring was continued at 0° C. with exclusion of light for 5 h. Thereaction was followed by HPLC. The brown reaction mixture was poured atonce into 550 ml water of 10° C. Over 30 min, 50 ml HCl 1N was added.The pH dropped from 4.6 to 3.2 and the product precipitated from thereaction mixture. Stirring was continued for 1 h at 0° C. The suspensionwas filtered. The product was washed with ice-cold water, re-suspendedin water, stirred for 20 min at room temperature, filtered and againwashed with water. (6R,7R)-7-(Z)-2-(2-aminothiazol-4-yl)-2-acetoxyimino-acetylamino)!-3-(E)-1-cyclopropylmethyl-2-oxo-pyrrolidin-3-ylidenemethyl!-8-oxo-5-thia-1-azabicyclo4.2.0!oct-2-ene-2-carboxylic acid was obtained as a beige, wet solid.The product was used immediately and without drying for the next step.

EXAMPLE 3

a) Preparation of (Z)-(RS)-(2-aminothiazol-4-yl)-(tetrahydropyran-2-yloxyimino)!-acetic acid diethoxythio-phosphorylester ##STR13##

To a stirred suspension of 30 g (Z)-(RS)-(2-aminothiazol-4-yl)-(tetrahydropyran-2-yloxyimino)!-acetic acid (80.5 mmol) and 90 mg1,4-diazabicyclo 2.2.2!octane (DABCO) (0.80 mmol) in 300 mldimethylacetamide was added under argon over 45 min 17 ml diethylchlorothiophosphate (104.9 mmol). Stirring was continued at 0° C. for 1h. The reaction was followed by HPLC. To the slightly turbid reactionmixture was added dropwise over 50 min 450 ml water. The precipitatedproduct was filtered, washed with water and dissolved indichloromethane. The aqueous layer was back-extracted withdichloromethane. The combined organic layers were dried over sodiumsulfate and concentrated under reduced pressure until the productstarted to crystallize. To the residual solution was added dropwise over30 min n-hexane. The resulting suspension was cooled to 2° C., stirredfor 1 h and then filtered. The crystalline product was washed withn-hexane and dried to constant weight. (Z)-(RS)-(2-aminothiazol-4-yl)-(tetrahydropyran-2-yloxyimino)!-acetic acid diethoxythio-phosphorylester was obtained as a white solid (28.01 g, yield=82%) and was storedunder argon at -20° C. No further purificiation was necessary and theproduct was used as isolated for the next step.

IR (KBr) 3423, 3261, 3169, 3145, 2946, 1772, 1614, 1541. 1388, 1241,1204, 1156, 1110, 1020, 973, 944, 908, 888, 857, 827, 727, 692; ¹ H-NMR(250 MHz, CDCl₃) δ 1.37 (t,J=7.1,6H); 1.50-1.95 (m,6H); 3.65(dm,J=11.4,1H); 3.86 (tm,J=11.4,1H); 4.33 (dq,J₁ =8.0,J₂ =7.0,4H); 5.47(s,br,1H); 6.56 (s,br,2H); 6.79 (s,1H); ³¹ P-NMR (100 MHz, CDCl₃) δ59.33; ISP-MS 446.4 (19, M+Na!⁺), 424.5 (26, M+H!⁺), 340.2 (100); MAcalculated for C₁₄ H₂₂ N₃ O₆ PS₂ C 39.71, H 5.24, N 9.92, S 15.14, P7.31; found C 39.87, H 5.20, N 10.08, S 14.99, P 7.53.

b) (6R,7R)-7- (Z)-2-(2-Aminothiazol-4-yl)-2-(R,S)-tetrahydropyran-2-yloxyiminoacetylamino)!-3-(E)-1-cyclopropylmethyl-2-oxo-pyrrolidin-3-ylidenemethyl!-8-oxo-5-thia-1-aza-bicyclo4.2.0!oct-2-ene-2-carboxylic acid ##STR14##

Under argon atmosphere to a stirred suspension of 20 g(E)-(6R,7R)-7-Amino-3-(1-cyclopropylmethyl-2-oxo-pyrrolidin-3-ylidenemethyl)-8-oxo-5-thia-1-azabicyclo4.2.0!oct-2-ene-2-carboxylic acid (57.2 mmol) in 140 mldimethylformamide was added 16 ml triethylamine (114.8 mmol) at 10° C.After 10 min, to the solution was cooled to 0° C. and 24.72 g(Z)-(RS)-(2-arinothiazol-4-yl)- (tetrahydropyran-2-yloxyinino)!-aceticacid diethoxythio-phosphoryl ester (58.4 mmol) was added in portionsover 1 min. Stirring was continued at 10° C. with the exclusion of lightfor 6 h. The reaction was followed by HPLC. The reaction mixture waspoured at once into a 10° C. mixture of 220 ml water and 50 ml acetone.Over 30 min. 55 ml HCl 1N was added. The pH dropped from 9.6 to 3.2 andthe product precipitated from the reaction mixture. Stirring wascontinued for 30 min at 0° C. The suspension was filtered. The productwas washed with ice-cold water and dried to constant weight. (6R,7R)-7-(Z)-2-(2-Aminothiazol-4-yl)-2-(R,S)-tetrahydropyran-2-yloxyimino-acetylamino)!-3-(E)-1-cyclopropylmethyl-2-oxo-pyrrolidin-3-ylidenemethyl!-8-oxo-5-thia-1-aza-bicyclo4.2.0!oct t-2-ene-2-carboxylic acid was obtained as an off-white solid(27.7 g). The product was used as isolated for the next step.

¹ H-NMR (250 MHz, DMSO) δ 0.21 (m,2H); 0.46 (m,2H); 0.93 (m,1H);1.40-1.90 (m,6H); 2.90-3.10 (m,2H); 3.16 (d,J=7.1,2H); 3.48 (m,2H); 3.50(m,1H); 3.85 (m,1H); 3.90 (s,2H); 5.21 (d,J=5.0,1H): 5.26 (s,br,1H):5.90 (dd,J₁ =8.2,J₂ =5.0, 1H); 6.75 (s,1H); 7.23 (s,br,3H); 9.69(d,J=8.2,1H); 13.95 (s,br,1H).

EXAMPLE 4

Cleavage of the Protective Groups:

Preparation of (6R,7R)-7-(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyimino-acetylamino!-3-(E)-1-cyclopropylmethyl-2-oxo-pyrrolidin-3-ylidenemethyl!-8-oxo-5-thia-1-aza-bicyclo4.2.0!oct-2-ene-2-carboxylic acid ##STR15## a) By cleavage of the tritylgroup

To a stirred solution of 30 g (6R, 7R)-7-(Z)-2-(2-Aminothiazol-4-yl)-2-trityloxyiminoacetylamino)!-3-(E)-1-cyclopropylmethyl-2-oxo-pyrrolidin-3-ylidenemethyl!-8-oxo-5-thia-1-aza-bicyclo4.2.0!oct-2-ene-2-carboxylic acid triethylanimonium salt (29.2 mmol) in60 ml dichloromethane was added over 15 min 7.5 ml triethylsilane (45.9mmol) and over 90 min 23.9 ml trifluoroacetic acid (306 mmol) at 2° C.Stirring was continued at 10° C. for 2 h. The reaction was followed byHPLC. To the reaction mixture was added over 90 min 300 ml diethylether,causing the product to precipitate. Stirring was continued for 1 h atroom temperature. The suspension was filtered. The product was washedwith 2×60 ml diethylether, again suspended in 100 ml diethylether,stirred for 15 min, filtered, washed with 2×40 ml diethylether and driedto constant weight. The trifluoroacetate of (6R,7R)-7-(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyimino-acetylamino!-3-(E)-1-cyclopropylmethyl-2-oxo-pyrrolidin-3-ylidenemethyl!-8-oxo-5-thia-1-aza-bicyclo4.2.0!oct-2-ene-2-carboxylic acid was obtained as an off-white solid(19.92 g, 99%, HPLC=100 area %) and suspended in 400 ml water. Over 15min 20 ml NaOH 1N (20 mmol) were added at 2° C. The pH rose from 1.51 to3.30. The suspension was stirred at 2° C. for 10 min and then filtered.For the filtration a mild vacuum of about 400 mbar was applied. Theproduct was washed with 2×50 ml water, suspended in 250 ml water,stirred for 15 min at 2° C., filtered, washed with 2×50 ml water andre-suspended in 400 ml water. Over 40 min 30 ml NaOH 1N was added at 2°C. The pH rose from 2.38 to 5.6 and most of the product dissolved. Theturbid solution was filtered and two membrane filters of 0.45 μm and0.22 μm. To the resulting, clear solution was added over 20 min 26 mlHCl 1N (26 mmol) at 2° C. The pH dropped from 5.42 to 3.30 and theproduct precipitated. The suspension was stirred for 60 min at 2° C.,filtered and washed with 100 ml water. The product was dried (15 mbar,24 h, 35° C.) to constant weight. (6R,7R)-7-(Z)-2-(2-Aminothiazol-4-yl)-2-hydroxyimino-acetylamino!-3-(E)-1-cyclopropylmethyl-2-oxo-pyrrolidin-3-ylidenemethyl!-8-oxo-5-thia-1-aza-bicyclo4.2.0!oct-2-ene-2-carboxylic acid was isolated as an off white solid(12.1 g, yield 81%, HPLC 94 area %).

¹ H-NMR (250 MHz, DMSO) δ 0.21 (m,2H), 0.46 (m,2H); 0.93 (m,1H); 2.90(m,1H); 3.10 (m,1H); 3.15 (d,J=7.0,2H); 3.48 (t,J=6.0,2H); 3.88 (s,2H);5.18 (d,J=4.9,1H); 5.82 (dd,J₁ =8.7,J₂ =4.9,1H); 6.66 (s,1H); 7.14(s,br,2H); 7.22 (s,1H); 9.51 (d,J=8.7,1H); 11.33 (s,br,1H).

Anal.

calculated for C₂₁, H₂₂, N₆ O₆ S₂ : C 48.64, H 4.28, N 16.21, S 12.36;found C 47.88, H 4.36, N 15.85, S 12.17 and 2.47% H₂ O.

b) By cleavage of the acetyl group

To a stirred suspension of (6R,7R)-7-(Z)-2-(2-aminothiazol-4-yl)-2-acetoxyimino-acetylamino)!-3-(E)-1-cyclopropylmethyl-2-oxo-pyrrolidin-3-ylidenemethyl!-8-oxo-5-thia-1-azabicyclo4.2.0!oct-2-ene-2-carboxylic acid (used in wet form, assumed ˜73.3 mmol)in 300 ml methanol was added under an argon atmosphere over 10 min 30 mlHCl conc. (304 mmol) at 2° C. After 5 h stirring at 2° C., another 10 mlHCl conc. (101 mmol) were added to the suspension. The reaction mixturewas allowed to warm up to room temperature over night. The reaction wasfollowed by HPLC. After 21 h total reaction time, all starting materialwas consumed and a brown solution had resulted. The reaction mixture waspoured at once into 800 ml ice cold water. To the resulting suspensionwas added over 60 min 500 ml NaOH 1N. The pH rose from 0.6 to 3.3.Stirring at 2° C. was continued for 15 min. The suspension was filtered.The product was washed with water and dried to constant weight.(6R,7R)-7- (Z)-2-(2-Aminothiazol-4-yl)-2-hydroxyimino-acetylamino!-3-(E)-1-cyclopropylmethyl-2-oxo-pyrrolidin-3-ylidenemethyl!-8-oxo-5-thia-1-aza-bicyclo4.2.0!oct-2-ene-carboxylic acid was obtained as a yellowish solid (29.8g, yield 78%, HPLC 90 area %).

c) By cleavage of the tetrahydropyranyl (THP) group

To a stirred suspension of 20 g, (6R,7R)-7-(Z)-2-(2-aminothiazol-4-yl)-2-(R,S)-tetrahydropyran-2-yloxyimino-acetylamino)!-3-(E)-1-cyclopropylmethyl-2-oxo-pyrrolidin-3-ylidenemethyl!-8-oxo-5-thia-1-aza-bicyclo4.2.0!oct-2-ene-2-carboxylic acid (33.3 mmol) in 150 ml methanol wasadded over 10 min 15 ml HCl conc. (180 mmol) at room temperature. Theyellow solution was stirred at 45° C. for 4.5 h. The reaction wasfollowed by HPLC. After 4 h all starting material was consumed. Thereaction mixture was allowed to cool to room temperature and poured atonce into 500 ml water. To the solution was added over 40 min 170 mlNaOH 1N. The pH rose from 0.43 to 3.1. The resulting suspension wascooled to 2° C., stirred for 1 h and filtered. The product was washedwith ice cold water and dried to constant weight. (6R,7R)-7-(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyimino-acetylamino!-3-(E)-1-cyclopropylmethyl-2-oxo-pyrrolidin-3-ylidenemethyl!-8-oxo-5-thia-1-aza-bicyclo4.2.0!oct-2-ene-2-carboxylic acid was obtained as a yellowish solid(12.8 g, yield 74%, HPLC 85 area %).

What is claimed is:
 1. A compound of formula ##STR16## wherein R¹ is trityl, acetyl, tetrahydropyranyl or cyclopentyl and R³ is lower alkyl.
 2. The compound of claim 1 wherein R¹ is trityl and R³ is ethyl.
 3. The compound of claim 1 wherein R¹ is acetyl and R³ is ethyl.
 4. The compound of claim 1 wherein R¹ is tetrahydropyranyl and R³ is ethyl. 